Aromatase activator

ABSTRACT

Drugs and cosmetics which are highly safe, and which are effective in preventing, improving, or treating various pathological conditions caused by estrogen depletion are provided. Provided are aromatase activators containing at least one member selected from plants such as Labiatae sps. including  Isodon, Scutellaria  (huangcen),  Schizonepeta  (jingjie), sage, lavender,  Lamium album , and thyme, an extract thereof; yeast extract; silk protein extract; milk protein; trehalose; natto extract; royal jelly;  oryza  oil; hydrolyzated wheat extract; shea butter; and rice fermentation extract.

FIELD OF THE INVENTION

This invention relates to an aromatase activator for increasing activityof aromatase which is an enzyme involved in biosynthesis of estrogenfrom androgen.

BACKGROUND OF THE INVENTION

Estrogen which is known to be a female sex hormone is produced in humanmainly by ovary, and known types include 17β-estradiol, estrone, andestriol.

Estrogen is involved in various physiological functions includingpropagation of endometrium, regulation of sexual functions, regulationof bone metabolism, and regulation of lipid metabolism, and therefore,estrogen depletion caused by aging or weakening of ovarian functionresults in symptoms such as climacteric disturbance, hypogonadism,autonomic imbalance, lipidosis, vasomotor disturbance, and osteoporosis.

In the meanwhile, prevention or improvement of such symptoms by directadministration of estrogen or estrogenic substance would beinappropriate since they have EDC (endocrine disrupting chemical)action.

SUMMARY OF THE INVENTION

This invention provides an aromatase activator containing at least onemember selected from the group consisting of Labiatae spp. which areIsodon, Scutellaria (huangcen), Schizonepeta (jingjie), sage, lavender,Lamium album, and thyme; Umbelliferae spp. which are fennel (huixiang),cnidium (chuangong), glehnia, angelica (danggui), bupleurum (chaihu),Saposhnikovia (fang feng), and angelica (baizhi); Rutaceae spp. whichare bitter orange (zhishi), Evodia (wuzhuyu), zanthoxylum, tangerine(chenpi), bitter orange (toupi), lemon, and grapefruit; Compositae spp.which are lettuce, Roman chamomile, arnica, Atractylodes (bai zhu),safflower, and yarrow; Leguminosae spp. which are liquorice (gancao),Sophora (kushen), restharrow, tragacanth, and cassia (juemingzi);Rosaceae spp. which are hawthorn (shanzhazi), apple, burnet, andwhitethorn; Zingiberaceae spp. which are turmeric (yujin), zedoary(woshu), cardamom, and ginger (shengjiang); Moraceae spp. which aremulberry (sangbaipi) and hop; Liliaceae spp. which are butcher's broomand lily; Gentianaceae spp. which are gentian (longdan) and gentian;Gramineae spp. which are sasa and imperata; Iridaceae sp which is iris(iris root); Lauraceae sp which is cinnamon; Juglandaceae sp which isEngelhardtia; Asclepiadaceae sp which is condurango; an Aristolochiaceaesp which is asiasarum (xixin); Dioscoreaceae sp which is dioscorea(shanyao); Acoraceae sp which is sweet flag; Betulaceae sp which isbirch; Caprifoliaceae sp which is honeysuckle (rendong); Myrtaceae spwhich is cloves; Hamamelidaceae sp which is hamamelis; Menispermaceae spwhich is Sinomenium (fangyi); Ephedraceae sp which is ephedra herb(mahuang); Ganodermataceae sp which is ling zhi; Hydrangeaceae sp whichis sweet hydrangeae; Papaveraceae sp which is corydalis (yanhusuo);Bignoniaceae sp which is catalpa; Magnoliaceae sp which is magnolia(houpu); Malvaceae sp which is mallow; Solanaceae sp which is tomato;Cucurbitaceae sp which is luffa; Pinaceae sp which is rosin; andTyphaceae sp. which is reed mace; an extract thereof; yeast extract;silk protein extract; milk protein; trehalose; natto extract; royaljelly; oryza oil; hydrolyzated wheat extract; shea butter; and ricefermentation extract.

This invention also provides use of the plant or the extract thereof,yeast extract, silk protein extract, milk protein, trehalose, nattoextract, royal jelly, oryza oil, hydrolyzed wheat extract, Shea butter,or rice fermentation extract as described above for producing anaromatase activator.

This invention also provides a method for activating aromatase includingadministering the plant or the extract thereof, yeast extract, silkprotein extract, milk protein, trehalose, natto extract, royal jelly,oryza oil, hydrolyzed wheat extract, Shea butter, or rice fermentationextract as described above to a human individual.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to provision of a drug or a cosmetic which ishighly safe, and which is effective in preventing, improving, ortreating various conditions as described above caused by estrogendepletion through promotion of estrogen production in the body.

The inventors of the present invention focused attention on biosynthesisof androgen from estrogen by aromatase, and investigated naturalsubstances that are capable of enhancing aromatase activity. Theinventors then found that particular plants and algae exhibit aromataseactivating actions.

The aromatase activator of the present invention is capable of promotingestrogen synthesis in the body, and is also highly safe to human body.Therefore, its use as a drug or a cosmetic for preventing, improving, ortreating various pathological conditions caused by estrogen depletion isquite effective.

In the aromatase activator of the present invention, Isodon means Isodonjaponicus or I. trichocarpus in the family Labiatae; Scutellaria(huangcen) means Scutellaria baicalensis in the family Labiatae;Schizonepeta (jingjie) means Schizonepeta tenuifolia in the familyLabiatae; sage means Salvia officinalis in the family Labiatae; lavendermeans Lavandula angustifolia in the family Labiatae; Lamium means Lamiumalbum in the family Labiatae; thyme means Thymus vulgaris the familyLabiatae; fennel (huixiang) means Foeniculum vulgare in the familyUmbelliferae; cnidium (chuangong) means Cnidium officinale in the familyUmbelliferae; glehnia means Glehnia littoralis in the familyUmbelliferae: angelica (danggui) means Angelica acutiloba in the familyUmbelliferae; bupleurum (chaihu) means Bupleurum falcatum in the familyUmbelliferae; Saposhnikovia (fang feng) means Saposhnikovia divaricataor Ledebouriella seseloides in the family Umbelliferae; angelica(baizhi) means Angelica dahurica in the family Umbelliferae; bitterorange (zhishi) means Citrus aurantium in the family Rutaceae; Evodia(wuzhuyu) means Evodia rutaecarpa or C. officinalis in the familyRutaceae; zanthoxylum means Zanthoxylum piperitum in the familyRutaceae; tangerine (chenpi) means Citrus unshiu in the family Rutaceae;bitter orange (toupi) means Citrus aurantium in the family Rutaceae;lemon means Citrus limon in the family Rutaceae; grapefruits meansCitrus paradisi in the family Rutaceae; lettuce means Lactuca sativa inthe family Compositae; Roman chamomile means Anthemis nobilis in thefamily Compositae; arnica means Arnica montana in the family Compositae;Atractylodes (bai zhu) means Atractylodes japonica or A. ovata in thefamily Compositae; safflowers means Carthamus tinctorius in the familyCompositae; yarrow means Achillea millefolium in the family Compositae;liquorice (gancao) means Glycyrrhiza glabra, G. uralensis or G. inflatain the family Leguminosae; Sophora (kushen) means Sophora angustifoliain the family Leguminosae; restharrow means Ononis spinosa in the familyLeguminosae; tragacanth means Astragalus gummifer in the familyLeguminosae; cassia (juemingzi) means Cassia obtusifolia in the familyLeguminosae; hawthorn (shanzhazi) means Crataegus cuneata in the familyRosaceae; apple means Pyrus malus in the family Rosaceae; burnet meansSanguisorba officinalis in the family Rosaceae; whitethorn meansCrataegus oxyacantha in the family Rosaceae; turmeric (yujin) meansCurcuma longa in the family Zingiberaceae; zedoary (woshu) means Curcumazedoaria in the family Zingiberaceae; cardamom means Elettariacardamomum in the family Zingiberaceae; ginger (shengjiang) meansZingiber officinale in the family Zingiberaceae; mulberry (sangbaipi)means Morus alba or M. bombycis in the family Moraceae; hop meansHumulus lupulus in the family Moraceae; butcher's broom means Ruscusaculeatus in the family Liliaceae; lily means Lilium candidum in thefamily Liliaceae; gentian (longdan) means Gentiana scabra in the familyGentianaceae; gentian means Gentiana lutea in the family Gentianaceae;sasa means Sasa Veitchii in the family Gramineae; imperata meansImperata cylindrica in the family Gramineae; iris (iris root) means Irisflorentina in the family Iridaceae; cinnamon means Cinnamomum cassia inthe family Lauraceae; Engelhardtia means Engelhardtia chrysolepis in thefamily Juglandaceae; condurango means Marsdenia cundurango in the familyAsclepiadaceae; asiasarum (xixin) measn Asarum sieboldii or A.heterotropoides in the family Aristolochiaceae; dioscorea (shanyao)means Dioscorea japonica in the family Dioscoreaceae; sweet flag meansAcorus calamus in the family Acoraceae; birch means Betula platyphyllaSuk. var. japonica in the family Betulaceae; honeysuckle (rendong) meansLonicera japonica in the family Caprifoliaceae; cloves means Syzygiumaromaticum in the family Myrtaceae; hamamelis means Hamamelis virginianain the family Hamamelidaceae; Sinomenium (fangyi) means Sinomeniumacutum in the family Menispermaceae; ephedra herb (mahuang) meansEphedra sinica in the family Ephedraceae; ling zhi means Ganodermalucidum in the family Ganodermataceae; sweet hydrangeae means Hydrangeaserrata in the family Hydrangeaceae; corydalis (yanhusuo) meansCorydalis turtschaninovii in the family Papaveraceae; catalpa meansCatalpa ovata in the family Bignoniaceae; magnolia (houpu) meansMagnolia obovata in the family Magnoliaceae; mallow means Malvasylvestris in the family Malvaceae; tomato means Lycopersicon esculentumthe family Solanaceae; Luffa means Luffa cylindrica in the familyCucurbitaceae; rosin means Pinus spp. in the family Pinaceae; and reedmace means Typha latifolia L., T. orientalis or T. augustifolia in thefamily Typhaceae.

Yeast extract is an extract produced from a solution obtained byautodigestion or acid-catalyzed hydrolysis of a Saccharomyces yeast, andexemplary commercially available products include the one sold under theproduct name of “Yeast Liquid B” (Ichimaru Pharcos Co Ltd.). Silkprotein extract is an extract obtained by acid-catalyzed hydrolysis ofsilk protein, and exemplary commercially available products include“Silkgen G Soluble KE” (Ichimaru Pharcos Co Ltd.). Milk proteins includelactose protein, lactoferrin, and the like, and exemplary commerciallyavailable products include “Bioderma SX-14” (Ichimaru Pharcos Co Ltd.)and “Lactoferrin S FREE” (Ichimaru Pharcos Co Ltd.). Trehalose istrehalose (molecular formula, C₁₂H₂₂O₁₁) and a typical products is“Trehalose” (Hayashibara) Natto extract is an extract produced byextracting natto (fermented soy bean) produced by fermentation ofsoybeans (Glycine max) with Bacillus subtilis, and exemplarycommercially available products include “Daizu Polymer F B-20” (IchimaruPharcos Co Ltd.). Royal jelly is an extract obtained from the substancesecreted by European honeybee Apis mellifica or Asian honeybee Apisindica, and an exemplary commercially available product is “Royal jellyextract” (Ichimaru Pharcos Co Ltd.).

Oryza oil is an oil produced from rice bran of rice grains, and anexemplary commercially available product is “Oryza oil S-1” (IchimaruPharcos Co Ltd.). Shea butter is a fat obtained from shea seeds, and anexemplary commercial available product is “Liquid Shea butter” (IchimaruPharcos Co Ltd.). Rice fermentation extract is an extract produced fromrice (Oryza sativa), and in particular, from the seeds coat of the rice,and an exemplary commercial available product is “rice fermentationextract” (Ichimaru Pharcos Co Ltd.). Hydrolyzed wheat extract is awater-soluble product obtained by hydrolyzing wheat (Triticum aestivum)flour, and an exemplary commercial available product is “Gluadin AGP”(Ichimaru Pharcos Co. Ltd.).

The plants as described above may be used either directly or withpulverization, and the part used may be whole plant, leaves, bark,twigs, fruits, roots, or the like. The preferable part used is:above-ground part for Isodon; roots for Scutellaria (huangcen);above-ground part or spikes for Schizonepeta (jingjie); leaves for sage;flowers for lavender; flowers for Lamium album; above ground part forthyme; mature fruits for fennel (huixiang); roots for cnidium(chuangong); roots and rhizomes for glehnia; roots for angelica(danggui); roots for bupleurum (chaihu); roots for Saposhnikovia (fangfeng); roots for angelica (baizhi); immature fruits for bitter orange(zhishi); fruits for Evodia (wuzhuyu); fruits for zanthoxylum; pericarpfor tangerine (chenpi); pericarp for bitter orange (toupi); fruits forlemon; fruits for grapefruits; leaves for lettuce; flowers for Romanchamomile; flowers for arnica; rhizomes for Atractylodes (bai zhu);flowers for safflowers; capitula for yarrow; roots for liquorice(gancao); roots for Sophora (kushen); roots for restharrow; materialssecreted from trunk for tragacanth; seeds for cassia; fruits forhawthorn (shanzhazi); fruits for apple; roots for burnet; fruits forwhitethorn; rhizomes for turmeric (yujin); rhizomes for zedoary (woshu);fruits for cardamom; rhizomes for ginger (shengjiang); roots bark formulberry (sangbaipi); Spikes for hop; roots for Butcher's broom; bulbsfor lily; roots for gentian (longdan); roots for gentian; leaves forsasa; rhizomes after removing rootslets and scaly leaves for imperata;roots for iris; bark for cinnamon; leaves for Engelhardtia; bark forcondurango; roots for asiasarum (xixin); rhizomes after removingdioscorea (shanyao); roots for sweet flag; bark for birch; flowers forhoneysuckle (rendong); flower bud for cloves; leaves for hamamelis; stemand rhizomes for Sinomenium (fangyi); terrestrial stem for ephedra herb(mahuang); fruit bodies for ling zhi; leaves for sweet hydrangeae;tubers for corydalis (yanhusuo); pericarp for catalpa, bark for magnolia(houpu); flowers for mallow; fruits for tomato; fruits for Luffa; resinremaining after removing essential oil from the secreted material forrosin; and spikes for reed mace.

In the present invention, the term extract includes various extractsobtained by extracting the plant as described above at room temperatureor at elevated temperature with or without using an extraction apparatussuch as Soxhlet extraction apparatus, a dilution and a concentratethereof, and a powder produced by drying the extract.

The extraction solvent used extracting the plants of the presentinvention may be either a polar solvent or a non-polar solvent.Exemplary solvents include water; methanol, ethanol, propanol, butanoland other alcohols; propylene glycol, butylene glycol, and otherpolyhydric alcohols; acetone, methyl ethyl ketone, and other ketones;methyl acetate, ethyl acetate, and other esters; tetrahydrofurane,diethylether, and other chain or cyclic ethers; polyethyleneglycol andother polyethers; squalane, hexane, cyclohexane, petroleum ether, andother hydrocarbons; toluene and other aromatic hydrocarbons;dichloromethane, chloroform, dichloroethane, and other halogenatedhydrocarbons; and carbon dioxide; and mixtures thereof.

The plant extract as described above may be used either with no furtherprocessing, or by diluting, concentrating, or freeze drying the extractand preparing a powder or paste.

Also, the plant or the extract thereof may be used after removinginactive contaminants from the extract by an adequate separationtechniques such as chromatography.

The plant or the extract thereof, yeast extract, silk protein extract,milk protein, trehalose, natto extract, royal jelly, oryza oil,hydrolyzed wheat extract, Shea butter and rice fermentation extract(hereinafter referred to as plants and the like) of the presentinvention may also be used as a mixture of two or more.

These plants and the like has the action of activating aromatase sincethey increase expression of the aromatase gene as will be demonstratedin the Examples. Therefore, when the aromatase activator containing theplants and the like incorporated in a drug or a cosmetic is administeredto a human individual, estrogen production in the body will be enhanced,and for this, the effects as described below owing to the estrogen areanticipated (Science of Body No. 219, 2001, Nihon Hyron-sha).

(1) Action on bone metabolism: The aromatase activator will suppressfunction of the parathyroid hormone, thereby suppressing boneresorption, and it will also activate vitamin D in kidney, therebysuppressing the progress of osteoporosis.

(2) Action on hyperlipidemia: The aromatase activator will preventdevelopment of atherosclerosis by the LDL accumulation in blood which isinduced by the decrease in the number of LDL receptor due to enhancementof LPL (lipoprotein lipase) activity by the decrease in estrogenconcentration. The aromatase activator will also increase expression ofmRNA in vascular endothelium to enhance NO production. The aromataseactivator will act to facilitate antioxidative action and vasodilatingaction while it will act to suppress arterial sclerosis.

(3) Action on brain function: The aromatase activator will improvecerebral function such as memory, cognitive function bring change incerebral blood flow, and influence on feeling and emotion. Relation todepression has also been reported. In the case of Alzheimer's disease,aromatase activator will (i) act on neurons to increase activity of Ach(acetylcholine) synthetase (choline acetyltransferase), (ii) stimulateexpression of receptors for nerve growth factor (NGF) and brain-derivedneurotrophic factor (BDNF) in cholinergic neuron, (iii) increase numberof synapses in hippocampus, (iv) ameliorate neuron damage by reducingthe accumulation of β-amyloid by acting on amyloid precursor protein(APP), and (v) improve sugar transportation and utilization in brain.

(4) Action on climacteric disturbance: The aromatase activator willimprove autonomic imbalance caused by hyperfunction of hypothalamus andhypophysis due to dysfunctioning of the negative feedback inhypothalamo-hypophysial-ovarian system caused by decrease of estrogen,that is, the autonomic imbalance caused by the increase of LH(luteinizing hormone) and FSH (follicle stimulating hormone).

(5) Action on eye: The aromatase activator will suppress onset ofmacular degeneration and cataracta which are popular in women afterclimacterium. It also improves function of lacrimal gland, suppressingdry eye.

When the aromatase activator of the present invention is incorporated ina drug, the drug may take the form of tablet, capsule or other oralmedicine, ointment, solution, extract, lotion, emulsion or otherexternal medicine, or injection.

When the aromatase activator of the present invention is incorporated ina cosmetic, the cosmetic may take various forms, for example,water-in-oil or oil-in-water emulsion, cream, lotion, gel, foam,essence, foundation, pack, stick, and powder. The cosmetic may alsocontain an oil, surfactant, UV absorber, alcohol, chelating agent, pHadjusting agent, antiseptic, thickener, colorant, perfume, skinnutrient, or other components commonly used as a component in thecosmetics in addition to the plant or its extract of the presentinvention.

Preferably, the plant and the like may be incorporated in the drug orthe cosmetic as described above at a content in dry basis of 0.00001 to1% by weight, preferably at 0.0001 to 0.1% by weight based on the totalweight.

EXAMPLES

Next, the present invention is described in further detail by referringto the following Examples.

Production Examples Preparation of Plant Extracts

Plant extracts as shown in Tables 1 and 2, below were prepared by theordinary method commonly used in the art.

TABLE 1 Residual content Extraction after Name of the Extract Part usedsolvent evaporation Sweet hydrangeae Leaves 50% EtOH 2.6 w/v % ArnicaFlowers 50% EtOH 0.9 w/v % Fennel (huixiang) Mature fruits 50% EtOH 4.5w/v % Turmeric (yujin) Rhizomes 50% EtOH 0.9 w/v % Corydalis (yanhusuo)Tubers 50% EtOH 4.0 w/v % Isodon Above ground 50% EtOH 1.0 w/v % partScutellaria Roots 50% EtOH 3.4 w/v % (huangcen) Zedoary (woshu) Rhizomes50% EtOH 1.7 w/v % Catalpa Pericarp 50% EtOH 4.1 w/v % Bitter orangeImmature fruits 50% EtOH 14.4 w/v %  (zhishi) Sasa Leaves 50% EtOH 0.8w/v % Schizonepeta Above ground 50% EtOH 1.7 w/v % (jingjie) part orspikes Cassia (juemingzi) Seed 50% EtOH 1.0 w/v % Magnolia (houpu) Bark50% EtOH 4.1 w/v % Evodia (wuzhuyu) Fruits 50% EtOH 11.6 w/v % Bupleurum (chaihu) Roots 50% EtOH 3.4 w/v % Asiasarum (xixin) Roots 50%EtOH 1.3 w/v % Zanthoxylum Fruits 50% EtOH 1.5 w/v % Cardamom Fruits 50%EtOH 2.5 w/v % Mallow Flowers 50% EtOH 0.5 w/v % Cnidium (chuangong)Roots 50% EtOH 3.6 w/v % Angelica (danggui) Roots 50% EtOH 4.2 w/v %Tomato Fruits water 1.0 w/v % Glehnia Roots and 50% EtOH 9.6 w/v %rhizomes Atractylodes (baizhu) Rhizomes 50% EtOH 8.0 w/v % Luffa Fruits50% EtOH 0.4 w/v % Safflower Flowers 95% EtOH 0.8 w/v % Reed mace Spikes50% EtOH 1.5 w/v % Lily Bulbs 50% EtOH 0.7 w/v % Gentian (longdan) Roots50% EtOH 13.1 w/v %  Rosin Resin after 50% EtOH 2.9 w/v % removal ofessential oil from the secreted material

TABLE 2 Residual content Extraction after Name of the extract Part usedsolvent evaporation Iris (iris root) Roots 50% EtOH 1.3 w/v % Lamiumalbum Flowers 50% EtOH 0.2 w/v % Restharrow Roots 50% EtOH 0.8 w/v %Reed mace Spikes 50% EtOH 1.5 w/v % Liquorice (gancao) Roots Water 2.4w/v % Sophora (kushen) Roots 50% EtOH 1.9 w/v % Grapefruits Fruits 50%EtOH 0.6 w/v % Cinnamon Bark 50% EtOH 0.8 w/v % Gentian Roots 50% EtOH4.2 w/v % Condurango Bark 50% EtOH 5.1 w/v % Asiasarum (xixin) Roots 50%EtOH 1.3 w/v % Sage Leaves 50% EtOH 2.4 w/v % Hawthorn Fruits 50% EtOH1.2 w/v % (shanzhazi) Dioscorea (shanyao) Rhizomes after 50% EtOH 2.4w/v % removing periderm Ginger (shengjiang) Rhizomes EtOH 0.5 w/v %Sweet flag Roots 50% EtOH 1.3 w/v % Birch Bark 50% EtOH 1.4 w/v %Honeysuckle Flowers 50% EtOH 0.6 w/v % (rendong) Whitethorn Fruits 50%EtOH 1.3 w/v % Yarrow Capitula 50% EtOH 0.4 w/v % Mulberry Roots bark50% EtOH 1.4 w/v % (sangbaipi) Thyme Above-ground 50% EtOH 2.7 w/v %part Cloves Flower buds 50% EtOH 2.2 w/v % Tangerine (chenpi) Pericarp50% EtOH 3.4 w/v % Bitter orange Pericarp 50% EtOH 3.7 w/v % (toupi)Tragacanth Materials 50% EtOH 7.4 w/v % secreted from trunk HamamelisLeaves 50% EtOH 0.2 w/v % Angelica Roots 50% EtOH 11.6 w/v %  (baizhi)Butcher's broom Roots 50% EtOH 1.2 w/v % Sinomenium (fangyi) Stems and50% EtOH 3.3 w/v % rhizomes Imperata Rhizomes after 50% EtOH 14.2 w/v %removing rootslets and scaly leaves Saposhnikovia Roots 50% EtOH 5.3 w/v% (fang feng) Hop Spikes Water 2.1 w/v % Ephedra herb Terrestrial 50%EtOH 5.9 w/v % (mahuang) stem Lavender Flowers 50% EtOH 2.1 w/v % AppleFruits 50% EtOH 8.0 w/v % Ling zhi Fruit bodies 50% EtOH 0.4 w/v %Lettuce Leaves 50% EtOH 0.3 w/v % Lemon Fruits 50% EtOH 0.6 w/v % Romanchamomile Flowers 50% EtOH 2.7 w/v % Burnet Roots 50% EtOH 2.2 w/v %

Referential Example 1 Construction of Reporter Gene Assay System

The region containing transcription control region for exon 1c of humanaromatase gene and a part of the exon 1c was amplified by PCR fromgenomic DNA extracted from human normal keratinocyte by using thefollowing primers:

Upper primer, 5′-GACTAGTAAACAACCACAAAACTGCTC-3′ (SEQ ID NO: 1) Lowerprimer, 5′-AACTGCAGACAAGTCAAAACAAGGAAGC-3′ (SEQ ID NO: 2)

The resulting PCR product was treated with restriction enzymes SpeI andPstI, and incorporated in SpeI site and PstI site in SeaPansy nullControl Vector (Toyo Ink Mfg. Co., Ltd.) to produce Ex1c-luc plasmid.This plasmid was used in the luciferase assay as will be describedbelow.

Example 1 Increase in the Expression of Exon 1c of Aromatase Gene (1)Materials and Methods

(i) Cells Used

Immortalized keratinocyte-derived cell (HaCaT cell)

(ii) Plasmid Used

About 1 kb of transcription control region for exon 1c of the aromatasegene was incorporated in the upstream of luciferase gene (Ex1c-luc).

(iii) Transfection into the Cell

HaCaT cell was propagated in a 100 mm dish to subconfluency, andEx1c-luc was introduced using lipofectamine reagent (Invitrogen)according to the protocol described in the attached manual. The DNA wasused at an amount of 8 μg per dish. The same procedure was repeated forthe control without adding the DNA (1 dish).

(iv) Luciferase Assay

The transfected cells were cultivated overnight, and inoculated to 96well cell culture plate at about 30,000 cells per well. Total amount ofthe culture medium was adjusted to be 200 μL. On the next day, plantextract prepared in Production Example 1, Table 1, or oryza oil (“oryzaoil S-1”, Ichimaru Pharcos Co Ltd.), Shea butter (“Liquid shea butter”,Ichimaru Pharcos Co Ltd.), yeast extract (“YeastLiquid B”, IchimaruPharcos Co Ltd.), rice fermentation extract (“rice fermentationextract”, Ichimaru Pharcos Co Ltd.), or hydrolyzed wheat extract(“Gluadin AGP”, Ichimaru Pharcos Co Ltd.) was added (1% or 0.1%), andthe cultivation was continued for another 20 hours. After adding 20 μLof alamarBlue (BIOSOURCE), fluorescence intensity (excitation light 544nm, fluorescence 590 nm) was measured. Luciferase activity was alsomeasured by using PicaGene Dual SeaPansy Luminescence kit (Nippon Gene).The cells were lysed by adding 25 μL per well of the lysis buffer thathad been diluted 5× lysis buffer to 1× concentration.

(2) Results

The results are shown in Table 3, below.

TABLE 3 Luciferase AlamarBlue activity activity Sweet hydrangeae 1%151.8% 89.1% Arnica 1% 121.3% 89.6% Fennel (huixiang) 0.1%   135.7%103.6% Fennel (huixiang) 1% 144.2% 110.0% Turmeric (yujin) 1% 402.2%116.2% Corydalis (yanhusuo) 0.1%   129.8% 90.0% Corydalis (yanhusuo) 1%135.3% 76.6% Isodon 1% 144.0% 113.9% Scutellaria (huangcen) 1% 163.6%62.8% Scutellaria (huangcen) 0.1%   139.2% 91.4% Phellodendron 1% 136.2%92.6% Oryza oil 0.1%   142.5% 85.5% Oryza oil 1% 134.0% 98.2% zedoary(woshu) 0.1% 125.4% 74.4% Catalpa 1% 142.2% 97.4% Bitter orange (zhishi)0.1%   135.5% 66.2% Bitter orange (zhishi) 1% 124.5% 64.8% Sasa 0.1%  128.8% 99.9% Hydrolyzed wheat extract 0.1%   121.3% 102.0% Schizonepeta(jingjie) 0.1%   125.1% 117.6% Schizonepeta (jingjie) 1% 177.5% 121.9%Cassia (juemingzi) 1% 127.1% 100.0% Magnolia (houpu) 0.1%   214.0%100.0% Evodia (wuzhuyu) 0.1%   140.2% 104.2% Bupleurum (chaihu) 1%139.5% 96.2% Asiasarum (xixin) 1% 134.7% 94.1% Zanthoxylum 1% 162.4%97.2% Cardamom 1% 162.4% 126.4% Mallow 1% 182.2% 93.6% Cnidium(chuangong) 0.1%   133.3% 99.8% Cndium (chuangong) 1% 172.5% 99.1%Angelica (danggui) 0.1%   122.0% 85.1% Angelica (danggui) 1% 175.1%87.9% Tomato 0.1%   127.2% 84.0% Tomato 1% 151.3% 79.9% Glehnia 1%121.4% 190.1% Atractylodes (bai zhu) 1% 123.2% 128.4% Luffa 0.1%  123.9% 90.8% Luffa 1% 138.2% 89.2% Safflower 0.1%   122.5% 104.8%Safflower 1% 160.8% 133.8% Reed mace 1% 122.2% 87.9% Lily 1% 122.3%121.9% Gentian (longdan) 1% 134.0% 159.4% Rosin 1% 140.2% 111.6% Sheabutter 0.1%   125.5% 95.9% Shea butter 1% 133.8% 104.1% Ricefermentation extract 1% 134.7% 98.1%

The results indicate that the extracts tested are capable of activatingaromatase expression.

Referential Example 2 Construction of Reporter Gene Assay System

The region containing transcription control region for exon 1b of humanaromatase gene and a part of the exon 1b was amplified by PCR fromgenomic DNA extracted from human normal keratinocyte by using thefollowing primers:

Upper primer, 5′-GACTAGTAAGGTGCAGTGACAGGCTC-3′ (SEQ ID NO: 3) Lowerprimer, 5′-GGAATTCCTGTCAGGCTCCAGTTGGTC-3′ (SEQ ID NO: 4)

The resulting PCR product was treated with restriction enzymes SpeI andEcoRI, and incorporated in SpeI site and EcoRI site in SeaPansy nullControl Vector (Toyo Ink Mfg. Co., Ltd.) to produce Ex1b-luc plasmid.This plasmid was used in the luciferase assay as will be describedbelow.

Example 2 Increase in the Expression of Exon 1b of Aromatase Gene (1)Materials and Methods

(i) Cells Used

Immortalized human hepatoma-derived cell (HepG2 cell)

(ii) Plasmid Used

About 1 kb of transcription control region for exon 1b of the aromatasegene was incorporated in the upstream of luciferase gene (Ex1b-luc).

(iii) Transfection into the Cell

HepG2 cell was inoculated to 96 well culture plate at 30,000 cells perwell, and Ex1b-luc was introduced using lipofectamine reagent(Invitrogen) according to the protocol described in the attached manual.The DNA was used at an amount of 0.1 μg per well. The same procedure wasrepeated for control without adding the DNA (2 wells).

(iv) Luciferase Assay

The transfected cells were cultivated overnight, and plant extractprepared in Production Example 1, Table 2, or yeast extract(“YeastLiquid B”, Ichimaru Pharcos Co Ltd.), silk protein extract(“Silkgen G Soluble KE”, Ichimaru Pharcos Co Ltd.), milk protein(“Bioderma SX-14” Ichimaru Pharcos Co Ltd.), “lactoferrin S FREE”,Ichimaru Pharcos Co Ltd.), natto extract (“Soybean polymer F B-20”,Ichimaru Pharcos Co Ltd.), or royal jelly (“royal jelly extract”,Ichimaru Pharcos Co Ltd.) was added (1% or 0.1%), and the cultivationwas continued for another 20 hours. After adding 20 μL of alamarBlue(BIOSOURCE), fluorescence intensity (excitation light 544 nm,fluorescence 590 nm) was measured. Luciferase activity was also measuredby using PicaGene Dual SeaPansy Luminescence kit (Nippon Gene). Thecells were lysed by adding 25 μL per well of the lysis buffer that hadbeen diluted 5× lysis buffer to 1× concentration.

(2) Results

The results are shown in Table 4, below.

TABLE 4 Luciferase AlamarBlue activity activity Iris (iris root) 0.1%  133.3% 120.2% Iris (iris root) 1% 202.5% 123.3% Lamium album 1% 123.2%91.5% Restharrow 0.1%   121.5% 102.5% Restharrow 1% 139.0% 103.4% Reedmace 1% 130.1% 124.3% Liquorice (gancao) 0.1%   122.0% 102.5% Silkprotein extract 0.1%   173.9% 170.3% Silk protein extract 1% 122.1%124.0% Lactose protein 0.1%   135.4% 109.1% Lactose protein 1% 133.5%129.0% Lactoferrin 0.1%   155.2% 135.9% Lactoferrin 1% 181.5% 138.2%Sophora (kushen) 0.1%   142.9% 160.1% Grapefruit 0.1%   120.7% 140.1%Grapefruit 1% 125.9% 132.7% Cinnamon 0.1%   121.7% 81.8% Gentian 0.1%  131.1% 123.9% Gentian 1% 122.4% 120.4% Engelhardtia 0.1%   130.5% 118.7%Engelhardtia 1% 156.9% 127.6% Yeast extract 1% 133.6% 83.4% Condurango0.1%   147.7% 107.2% Asiasarum (xixin) 0.1%   130.7% 122.1% Asiasarum(xixin) 1% 134.0% 118.9% Sage 0.1%   141.1% 92.7% Hawthorn (shanzhazi)1% 144.3% 100.1% Dioscorea (shanyao) 0.1%   121.4% 85.2% Ginger(shengjiang) 0.1%   123.1% 115.9% Sweet flag 1% 149.6% 73.6% Birch 1%139.8% 70.3% Honeysuckle (rendong) 0.1%   125.2% 84.1% Honeysuckle(rendong) 1% 135.1% 77.9% Whitethorn 0.1%   137.1% 84.9% Whitethorn 1%152.9% 77.1% Yarrow 1% 149.3% 67.9% Mulberry (sangbaipi) 1% 137.8% 90.1%Natto extract 0.1%   150.0% 108.0% Thyme 1% 135.5% 109.7% Cloves 1%175.2% 76.5% Tangerine (chenpi) 0.1%   131.5% 112.2% Trehalose 1% 141.0%113.9% Bitter orange (toupi) 1% 157.9% 119.5% Tragacanth 1% 121.5% 94.5%Hamamelis 1% 129.9% 126.0% Angelica (baizhi) 0.1%   247.7% 97.2%Butcher's broom 0.1%   132.8% 118.1% Sinomenium (fangyi) 0.1%   129.5%91.0% Sinomenium (fangyi) 1% 150.8% 82.9% Imperata 0.1%   123.6% 90.9%Saposhnikovia (fang feng) 0.1%   139.1% 106.6% Hop 0.1%   128.4% 102.2%Hop 1% 123.4% 109.2% Ephedra herb (mahuang) 0.1%   124.6% 87.8% Lavender1% 123.0% 95.1% Apple 1% 140.6% 100.5% Ling zhi 1% 166.0% 154.6% Lettuce1% 149.6% 133.9% Lemon 1% 146.1% 90.9% Roman chamomile 0.1%   137.5%178.4% Roman chamomile 1% 171.2% 175.1% Royal jelly 0.1%   120.1% 142.5%Burnet 0.1%   130.0% 83.0%

The results indicate that the extracts tested are capable of activatingaromatase expression.

1. A method of increasing expression of the aromatase gene comprisingtreating a cell population with an effective amount of an aromataseactivator comprising an ethanol extract of Iris florentina, wherein saideffective amount ranges from 0.0001 to 1% by weight based on the totalweight of the aromatase activator on a dry basis.
 2. The method of claim1, wherein the treating comprises administering the aromatase activatorto a human.
 3. The method of claim 2, wherein the aromatase activator isin a form of a cosmetic.
 4. The method of claim 2, wherein the aromataseactivator is in a form of a drug.
 5. The method of claim 2, wherein thearomatase activator is administered orally, is injected, or is topicallyadministered.
 6. The method of claim 1, wherein the ethanol extract ofIris florentina is an ethanol extract of roots of Iris florentina. 7.The method of claim 6 wherein the treating comprises administering thearomatase activator to a human.
 8. The method of claim 7, wherein thearomatase activator is in a form of a cosmetic.
 9. The method of claim7, wherein the aromatase activator is in a form of a drug.
 10. Themethod of claim 7, wherein the aromatase activator is administeredorally, is injected, or is topically administered.